Assay

ABSTRACT

This invention relates to truncated Tec kinase polypeptides and their use in screening for compounds which modulate the activity of Tec kinase polypeptides. Also described are nucleotide sequences encoding truncated Tec kinase polypeptides, vectors and host cells containing said nucleotides.

FIELD OF INVENTION

[0001] The present invention relates to truncated Tec kinasepolypeptides, nucleotide sequences encoding truncated Tec kinasepolypeptides, vectors and host cells containing said nucleotides,methods of screening for compounds which modulate the activity of Teckinase polypeptides, and the use of compounds identifiable by saidmethod in therapy.

BACKGROUND TO THE INVENTION

[0002] Antigen receptors on T, B and mast cells are multimolecularcomplexes that are activated by interactions with external signals.These signals are then transmitted to regulate gene expression andposttranscriptional modifications. A family of non-receptor tyrosinekinases known as the “Tec kinase family”, including Itk, Tec, Btk, Bmxand Txk tyrosine kinases, is involved in the signal transduction in T, Band mast cells. The members of the Tec kinase family share a similardomain structure, having an N-terminal pleckstrin-homology (PH) domain;a Tec homology domain (TH), which includes one (Itk, Bmx, Txk, andTec29) or two (Btk and Tec) proline-rich regions (PR); Src homology 3(SH3) and Src homology 2 (SH2) domains; and a catalytic kinase domain(SH1), see for exampie Yang et al. (2000) Immunity 12:373-382.

[0003] A need exists to identify modulators of Tec kinase polypeptidesin order to provide compounds for use in therapy. At present, there areno methods available for screening Tec kinase polypeptides. Accordingly,the object of the present invention is to provide a screen for compoundswhich modulate the activity of Tec kinase polypeptides and therebyprovide compounds for use in therapy.

SUMMARY OF INVENTION

[0004] The present invention is based on the finding that a truncatedTec kinase polypeptide can be used in an assay to screen for compoundswhich modulate the activity of Tec kinase polypeptides.

[0005] Accordingly, the invention provides a truncated Tec kinasepolypeptide having a Tec kinase amino acid sequence truncated by aminimum of the amino acids constituting the PH domain and a portion ofthe TH domain including at least one proline rich region up to but notincluding the amino acids constituting the kinase domain.

[0006] Another aspect of the invention is an isolated polynucleotidewhich (a) encodes a truncated Tec kinase polypeptide of the invention;(b) is complementary to polynucleotide (a); (c) selectively hybridisesto polynucleotide (a) or (b); or (d) is degenerate as a result of thegenetic code from polynucleotide (a), (b) or (c).

[0007] Further aspects of the invention are:

[0008] an expression vector which comprises a polynucleotide of theinvention and which is capable of expressing a polypeptide of theinvention;

[0009] a host cell comprising an expression vector of the invention;

[0010] a primer which is capable of generating a polynucleotide of theinvention;

[0011] a method of producing a polypeptide of the invention which methodcomprises introducing into an appropriate cell line a vector comprisinga polynucleotide of the invention under conditions suitable forobtaining expression of the polypeptide;

[0012] a method for the identification of a compound which modulates theactivity of a Tec kinase polypeptide, comprising contacting apolypeptide of the invention with a test compound and detecting anyenhancement or inhibition in the activity of the polypeptide, comparedto the activity that would occur in the absence of said test compound;

[0013] a compound which modulates Tec kinase activity and isidentifiable by the method referred to above;

[0014] use of a compound which modulates Tec kinase activity and isidentifiable by the method referred to above for the manufacture of amedicament for the treatment or prophylaxis of a disorder that isresponsive to modulation of Tec kinase activity; and

[0015] a method of treating a subject having a disorder that isresponsive to modulation of Tec kinase activity, which method comprisesadministering to said subject an effective amount of a compoundidentifiable by the method referred to above.

DETAILED DESCRIPTION OF THE INVENTION

[0016] We have surprisingly found that a truncated Tec kinasepolypeptide, which is truncated by a minimum of the amino acidsconstituting the PH domain and at least one of the proline rich regionof the TH domain, up to but not including the amino acids constitutingthe kinase domain, is suitable for screening for compounds whichmodulate the activity of Tec kinase polypeptides. The truncated Teckinase polypeptides of the present invention are constitutively activedespite truncation of a large portion of the protein.

[0017] The polypeptides of the invention have been found to beparticularly suitable for screening as they do not need to bepre-activated by phosphorylation. In vivo, Tec kinases need to bephosphorylated by other kinases in order to activate the enzyme (Gibsonet al. (1996) J. Immunology 156:2716-2722). Whilst pre-activation byphosphorylation is commonly required in assays, for example, LCK assaysinclude a phosphorylation step (Trevillyan, et al (1999) Arch. Biochem.Biophys. 364, 19-29), the removal of the need to preactivate thepolypeptides of the present invention offers a simplification for theassay.

[0018] A further advantage of the present invention is the provision ofan assay that is “robust”. The present inventors have found that theassay is robust in two respects. Firstly, it is possible to generatelarge amounts of truncated enzyme which are stable over a long time.Secondly, the assay gives a high frequency of comparable results uponrepeat testing.

[0019] The polypeptides of the invention may therefore provide usefulscreening targets for the identification and development of novelpharmaceutical agents, including agonists and antagonists of Teckinases, which may be useful in the treatment and/or prophylaxis ofdisorders such as inflammation.

[0020] Truncated Tec Kinase Polypeptides

[0021] The invention provides a truncated Tec kinase polypeptide havinga Tec kinase amino acid sequence truncated by a minimum of the aminoacids constituting the PH domain and a portion of the TH domainincluding at least one proline rich region up to but not including theamino acids constituting the kinase domain. In other words, the point oftruncation can occur anywhere after the first proline rich region of theTH domain (i.e. the proline rich region closest to the PH domain) andbefore the kinase domain. Preferably, the Tec kinase amino acid sequenceis truncated by a minimum of the amino acids constituting the PH domainand a portion of the TH domain including at least one proline richregion up to but not including the amino acids constituting the SH2 andkinase domain, i.e. the point of truncation can occur anywhere after thefirst proline rich region of the TH domain and before the SH2 domain.More preferably, the Tec kinase amino acid sequence is truncated by theamino acids constituting PH domain and a portion of the TH domainincluding at least one proline rich region up to but not including theamino acids constituting the SH3, SH2 and kinase domain, i.e. the pointof truncation can occur anywhere after the first proline rich region ofthe TH domain and before the SH3 domain. In a preferred aspect of theinvention, when the Tec kinase polypeptide contains more than oneproline rich region, the truncated Tec kinase polypeptide does notcontain any proline rich regions, i.e. the point of truncation occursafter all the proline rich regions of the TH domain.

[0022] The domains of the Tec kinase polypeptides referred to above aredefined in FIGS. 5 and 6. FIG. 6 shows a schematic representation of thedomain structure of Tec kinases.

[0023] The “PH domain” as described herein is the N-terminal domain ofthe Tec kinase polypeptide. It comprises β sheet structure.

[0024] The “TH domain” as described herein is the domain situatedbetween the PH and SH3 domains of the Tec kinase polypeptide. Itcomprises a globular core and either one (Itk, Bmx, Txk, Tec29) or two(Tec, Btk) proline rich (PR) regions.

[0025] The “proline rich regions” as described herein are the prolinerich regions in the TH domain. Preferably, a proline rich regioncomprises at least two proline residues within six consecutive aminoacids, preferably at least three proline residues within six consecutiveamino acids, more preferably at least four proline residues within sixconsecutive amino acids.

[0026] The “SH3 domain” as described herein is the domain situatedbetween the TH and SH2 domains of the Tec kinase polypeptide. Itcomprises two P sheets positioned at approximately right angles to eachother.

[0027] The “SH2 domain” as described herein is the domain situatedbetween the SH3 and kinase domain of the Tec kinase polypeptide.

[0028] The “kinase domain” as described herein is the C-terminal domainof the Tec kinase polypeptide. The “kinase domain” may alternatively beknown as the “SH1” domain.

[0029] A “Tec kinase polypeptide” as described herein is a polypeptideof the Tec kinase family, including Itk, Tec, Btk, Bmx and Txk tyrosinekinases.

[0030]FIG. 5 shows an alignment of the polypeptide sequences of Tecfamily kinases showing the PH, TH, SH3 and SH2 domains. The ClustaIXanalysis program was used to align the sequences (Thompson, J. D.,Gibson, T. J., Plewniak, F., Jeanmougin, F. and Higgins, D. G. (1997)The ClustalX windows interface: flexible strategies for multiplesequence alignment aided by quality analysis tools. Nucleic AcidsResearch, 24:4876-4882). The default settings were used.

[0031] Accordingly, in a preferred aspect of the invention, the Teckinase polypeptide is truncated at a position between amino acid 199(last amino acid of the first proline rich region of the TH domain, whenthe sequence is aligned as shown in FIG. 5) and amino acid 451 (firstamino acid of the kinase domain, when the sequence is aligned as shownin FIG. 5). More preferably the Tec kinase polypeptide is truncated at aposition between amino acid 199 (last amino acid of the first prolinerich region of the TH domain, when the sequence is aligned as shown inFIG. 5) and amino acid 417 (last amino acid of the SH2 domain, when thesequence is aligned as shown in FIG. 5). More preferably the Tec kinasepolypeptide is truncated at a position between amino acid 199 (lastamino acid of the first proline rich region of the TH domain, when thesequence is aligned as shown in FIG. 5) and amino acid 333 (first aminoacid of the SH2 domain, when the sequence is aligned as shown in FIG.5). Even more preferably the Tec kinase polypeptide is truncated at aposition between amino acid 199 (last amino acid of the first prolinerich region of the TH domain, when the sequence is aligned as shown inFIG. 5) and amino acid 224 (first amino acid of the SH3 domain, when thesequence is aligned as shown in FIG. 5).

[0032] In a more preferred aspect of the invention, when the Tec kinasepolypeptide has more than one proline rich region, the Tec kinasepolypeptide is truncated at a position between amino acid 213 (lastamino acid of the second proline rich region of the TH domain, when thesequence is aligned as shown in FIG. 5) and amino acid 451 (first aminoacid of the kinase domain, when the sequence is aligned as shown in FIG.5). More preferably the Tec kinase polypeptide is truncated at aposition between amino acid 213 (last amino acid of the second prolinerich region of the TH domain, when the sequence is aligned as shown inFIG. 5) and amino acid 417 (last amino acid of the SH2 domain, when thesequence is aligned as shown in FIG. 5). More preferably the Tec kinasepolypeptide is truncated at a position between amino acid 213 (lastamino acid of the second proline rich region of the TH domain, when thesequence is aligned as shown in FIG. 5) and amino acid 333 (first aminoacid of the SH2 domain, when the sequence is aligned as shown in FIG.5). Even more preferably the Tec kinase polypeptide is truncated at aposition between amino acid 213 (last amino acid of the second prolinerich region of the TH domain, when the sequence is aligned as shown inFIG. 5) and amino acid 224 (first amino acid of the SH3 domain, when thesequence is aligned as shown in FIG. 5).

[0033] In a particular aspect of the invention, the start of the SH3domain is defined by the amino acid sequence:*X-X-V-[VIK]-A-[LM]-Y-D-[YF]; the start of the SH2 domain is defined bythe amino acid sequence: [IL]-[ED]-X-Y-E-*W-Y; and the start of thekinase domain is defined by the amino acid sequence:G-[LF]-[GRS]-Y-[GDE]-[SK]-W-*, where,

[0034] the letters denote amino acids in one letter code,

[0035] the square brackets denote a single amino acid,

[0036] the amino acids within the square brackets are alternatives,

[0037] X is any one amino acid residue, and

[0038] “*” indicates the start of the domain.

[0039] For example, the Tec kinase polpeptide sequences to be truncatedmay be selected from Btk (human)—Accession no. Q06187 (SwissProt), Btk(mouse)—Accession no. P35991 (SwissProt), Itk (human)—Accession no.Q08881 (SwissProt), Itk (mouse)—Accession no. Q03526 (SwissProt), Tec(human)—Accession no. P42680 (SwissProt), Tec (mouse)—Accession no.P24604 (SwissProt), Bmx (human)—Accession no. P51813 (SwissProt), Bmx(mouse)—Accession no. P97504 (TREMBL), Txk (human)—Accession no. P42681(SwissProt), and Txk (mouse)—Accession no. P42682 (SwissProt).

[0040] Preferably the truncated Tec kinase polypeptide is a truncatedItk polypeptide or a truncated Btk polypeptide. Preferably the Itkpolypeptide to be truncated has a sequence as set forth in FIG. 4 (“ITK”sequence) or a homolog or variant thereof. Preferably the truncated ltkpolypeptide has the polypeptide sequence set forth in FIG. 3 or ahomolog or variant thereof. More preferably, the truncated Itkpolypeptide has a sequence encoded by the polynucleotide sequence setforth in FIG. 2. Preferably the Btk polypeptide to be truncated has asequence Btk (human) Accession no. 006187 (Swiss prot. Database) or ahomolog or variant thereof. Preferably, the truncated Btk polypeptidehas the polypeptide sequence set forth in FIG. 8 or a homolog or variantthereof. More preferably, the truncated Btk polypeptide has a sequenceencoded by the polynucleotide sequence set forth in FIG. 9.

[0041] The polypeptides of the present invention are provided in anisolated form. The term “isolated” is intended to convey that thematerial is not in its native state. Thus, the naturally-occurringpolypeptide present in a living animal is in its native state and is notisolated, but the same polypeptide, separated from some or all of thematerials it co-exists with in the natural system, is isolated. Thepolypeptides may be mixed with carriers or diluents which will notinterfere with their intended use and still be regarded as isolated.Similarly, a polypeptide which has been produced by synthetic means, forexample, by recombinant methods is “isolated”. The polypeptides of thepresent invention are also preferably provided in purified form andpreferably are purified to at least 50% purity, more preferably about75% purity, most preferably 90% purity or greater, such as 95%, 98%pure. Routine methods can be employed to purify and/or synthesize thepolypeptides according to the invention. Such methods are wellunderstood by persons skilled in the art, and include techniques such asthose disclosed in Sambrook et al, Molecular Cloning: a LaboratoryManual, 2^(nd) Edition, CSH Laboratory Press, 1989, the disclosure ofwhich is included herein in its entirety by way of reference.

[0042] The polypeptide of the present invention may be a recombinant ora synthetic polypeptide. The polypeptide of the invention is a human oranimal sequence (or homologous to such sequence). Such an animal istypically a mammal, such as a rodent (e.g. a mouse) or a primate.Preferably the polypeptide is a human sequence.

[0043] Homologues of polypeptide sequences are referred to above. Suchhomologues typically have at least 70% homology, preferably at least80%, 90%, 95%, 97% or 99% homology, for example over a region of atleast 15, 20, 30, 100 or more contiguous amino acids. The homology maybe calculated on the basis of amino acid identity (sometime referred toas “hard homology”). Identity is calculated using the widely used GCG(University of Wisconsin) suite of programs and preferably using thedistances software (correction method).

[0044] The term “variant” refers to a polypeptide which has a sameessential character or basic biological functionality as the truncatedTec kinase polypeptide in question. Preferably a variant polypeptide isone which binds to the same ligand as the truncated Tec kinasepolypeptide. Such variants may include allelic variants and thedeletion, modification or addition of single amino acids or groups ofamino acids within the polypeptide sequence.

[0045] Amino acid substitutions may be made, for example from 1, 2 or 3to 10, 15, 20 or 30 substitutions. The modified polypeptide retainsactivity as a truncated Tec kinase polypeptide. Changes in amino acidsequence of peptides can be guided by known similarities among aminoacids and other molecules or substituents in physical features such ascharge density, hydrophobicity, hydrophilicity, size and configurationetc. For example the amino acid Thr may be replaced by Ser and viceversa, and Leu may be replaced by lie and vice versa. For example, apolar amino acid such as glycine or serine may be substituted foranother polar amino acid; a basic amino acid may be substituted foranother basic amino acid; an acidic amino acid may be substituted foranother acidic amino acid; or a non-polar amino acid may be substitutedfor another non-polar amino acid. Groups of amino acids normallyconsidered to be equivalent are: (a) Ala (A), Ser (S), Thr (T), Pro (P),Gly (G); (b) Asn (N), Asp (D), Glu (E), Gln (Q); (c) His (H), Arg (R)Glu (E), Gln (Q); (d) Met (M), Leu (L), Ile (I), Val (V); and (e) Phe(F), Tyr (Y), Trp (W).

[0046] Polypeptides of the invention may be chemically modified, e.g.post-translationally modified. For example, they may be glycosylated orcomprise modified amino acid residues. They may also be modified by theaddition of histidine residues to assist their purification, or othersuitable protein tags, see for example, Nilsson et al. (1997) ProteinExpression and Purification 11:1-16. Such modified polypeptides fallwithin the scope of the term “polypeptide” of the invention.

[0047] PolynLicleotides and Primers

[0048] A further aspect of the invention is an isolated polynucleotidewhich (a) encodes a truncated Tec kinase polypeptide of the invention;(b) is complementary to polynucleotide (a); (c) selectively hybridisesto polynucleotide (a) or (b); or (d) is degenerate as a result of thegenetic code from polynucleotide (a), (b) or (c). A preferred aspect isan isolated polynucelotide which: (a) encodes the truncated ltkpolypeptide set forth in FIG. 3; (b) is complementary to polynucleotide(a); (c) selectively hybridises to polynucleotide (a) or (b); or (d) isdegenerate as a result of the genetic code from polynucleotide (a), (b)or (c). A more preferred aspect of the invention is an isolatedpolynucleotide having (a) the sequence set forth in FIG. 2; (b) asequence complementary to polynucleotide (a); (c) a sequence whichselectively hybridises to polynucleotide (a) or (b); or (d) a sequencethat is degenerate as a result of the genetic code from polynucleotide(a), (b) or (c). A further aspect of the invention, is an isolatedpolynucelotide which: (a) encodes the truncated Btk polypeptide setforth in FIG. 8; (b) is complementary to polynucleotide (a); (c)selectively hybridises to polynucleotide (a) or (b); or (d) isdegenerate as a result of the genetic code from polynucleotide (a), (b)or (c). A more preferred aspect of the invention is an isolatedpolynucleotide having (a) the sequence set forth in FIG. 9; (b) asequence complementary to polynucleotide (a); (c) a sequence whichselectively hybridises to polynucleotide (a) or (b); or (d) a sequencethat is degenerate as a result of the genetic code from polynucleotide(a), (b) or (c).

[0049] The polynucleotide sequences of the present invention may be inthe form of RNA or in the form of DNA, for example cDNA, genomic DNA,and synthetic DNA. Preferably the polynucleotide sequence of theinvention is cDNA. The DNA may be double-stranded or single-stranded,and if single stranded may be the coding strand or non-coding(anti-sense) strand.

[0050] As used herein the term polynucleotide includes nucleic acidsthat contain one or more modified (e.g., tritylated) or unusual (e.g.,inosine) bases. Thus, DNAs or RNAs with backbones modified for stabilityor for other reasons are polynucleotides as that term is intendedherein.

[0051] The polynucleotide of the invention is a human or animal sequence(or homologous to such sequence). Such an animal is typically a mammal,such as a rodent (e.g. a mouse) or a primate. Preferably thepolynucleotide is a human sequence.

[0052] Homologues of polynucleotide sequences typically have at least70% sequence identity, preferably at least 80%, 90%, 95%, 97% or 99%sequence identity, for example over a region of at least 15, 20, 30, 100or more contiguous nucleotides. Methods of measuring nucleic acidhomology are well known in the art. For example, the UWGCG Packageprovides the BESTFIT program which can be used to calculate homology(Devereux et al 1984). Similarly the PILEUP and BLAST algorithms can beused to line up sequences (for example are described in Altschul 1993,and Altschul et al 1990). In accordance with the invention, the defaultsettings may be used.

[0053] As used herein, “selective hybridisation” means that generallythe polynucleotide can hybridize to the gene region sequence at a levelsignificantly above background. The signal level generated by theinteraction between a polynucleotide of the invention and the generegion sequence is typically at least 10 fold, preferably at least 100fold, as intense as interactions between other polynucleotides and thegene region sequence. The intensity of interaction may be measured, forexample, by radiolabelling the polynucleotide, e.g. with ³²P. Selectivehybridisation may typically be achieved using conditions of lowstringency (0.3M sodium chloride and 0.03M sodium citrate at about 40°C.), medium stringency (for example, 0.3M sodium chloride and 0.03Msodium citrate at about 50° C.) or high stringency (for example, 0.03Msodium chloride and 0.003M sodium citrate at about 60° C).

[0054] The coding sequence of polynucleotides of the present inventionmay be modified by nucleotide substitutions, for example from 1, 2 or 3to 10, 15, 25, 50 or 100 substitutions. The polynucleotide mayalternatively or additionally be modified by one or more insertionsand/or deletions and/or by an extension at either or both ends. Themodified polynucleotide encodes a polypeptide which has the activityassociated with a truncated Tec kinase polypeptide. Degeneratesubstitutions may be made and/or substitutions may be made which wouldresult in a conservative amino acid substitution when the modifiedsequence is translated, for example as shown above.

[0055] Generation of a Tec kinase oligonucleotide may be performed bymethodologies known in the art such as polymerase chain reaction (PCR)for example on genomic DNA or cDNA with appropriate oligonucleotideprimers derived from or designed based on a knowledge of the sequence ofthe Tec kinase of interest. For example, the polynucleotide sequences ofTec kinases may be selected from: Btk (human)—Accession no. X58957(EMBL/GenBank), Btk (mouse)—Accession no. L08967 (EMBL/GenBank), Itk(human)—Accession no. D13720 (EMBL/GenBank), Itk (mouse)—Accession no.L00619 (EMBL/GenBank), Tec (human)—Accession no. D29767 (EMBL/GenBank),Tec (mouse)—Accession no. S53716 (EMBL/GenBank), Bmx (human)—Accessionno. X83107 (EMBL/GenBank), Bmx (mouse)—Accession no. U88091(EMBL/GenBank), Txk (human)—Accession no. L27071 (EMBL/GenBank), and Txk(mouse)—Accession no. U16145 (EMBL/GenBank).

[0056] According to a further aspect of the invention, there is provideda primer which is capable of generating a Tec kinase polynucleotidewhich encodes a truncated Tec kinase polypeptide of the invention. Anallele specific primer is used, generally together with a constantprimer, in an amplification reaction such as a PCR reaction, whichprovides discrimination between alleles through selective amplificationof one allele at a particular sequence position. The allele specificprimer is preferably at least 10, preferably at least 15 or at least 20,for example at least 25, at least 30 nucleotides in length. For example,in the generation of a truncated ltk polypeptide, the primers shown inFIG. 1 may be used to generate the corresponding polynucleotide. In thegeneration of a truncated Btk polypeptide, the primers shown in FIG. 7may be used to generate the corresponding polynucleotide.

[0057] The polynucleotide sequences of the present invention areprovided in an isolated form. The term “isolated” is intended to conveythat the material is not in its native state. Thus, thenaturally-occurring polynucleotide sequence present in a living animalis in its native state and is not isolated, but the same polynucleotidesequence, separated from some or all of the materials it co-exists within the natural system, is isolated. They may be mixed with carriers ordiluents which will not interfere with their intended use and still beregarded as isolated. Such polynucleotide sequence could be part of avector. Such polynucleotide'sequence could be part of a composition, andstill be isolated in that such vector or composition is not part of itsnatural environment. The polynucleotide sequences of the presentinvention are also preferably provided in purified form, and preferablyare purified to at least 50% purity, more preferably about 75% purity,most preferably 90% purity or greater, such as 95%, 98% pure.

[0058] Production of Tec Kinase Polypeptides

[0059] The polynucleotide sequences of the present invention may beemployed for producing a polypeptide of the invention by recombinanttechniques. Thus, for example the nucleotide sequence may be included inany one of a variety of expression vehicles or cloning vehicles, inparticular vectors or plasmids for expressing a protein.

[0060] A further aspect of the invention is therefore a vectorcomprising a polynucleotide of the invention. Appropriate cloning andexpression vectors for use with prokaryotic and eukaryotic hosts includemammalian expression vectors, insect expression vectors, yeastexpression vectors, bacterial expression vectors and viral expressionvectors, see Sambrook et al., Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor, N.Y., (1989). Examples of suitablevectors include derivatives of bacterial plasmids; phage DNA; yeastplasmids; vectors derived from combinations of plasmids and phage DNAand viral DNA and baculoviruses. A preferred vector is a baculovirus.

[0061] In a preferred aspect, the vector further comprises one or moreregulatory sequences to direct mRNA synthesis, including, for example, apromoter, operably linked to the sequence. Suitable promoters include:insect cell promoters, polyhedrin promotor, p10 and other promotersknown to control expression of genes in prokaryotic or eukaryotic cellsor their viruses. The vector may contain an enhancer and a ribosomebinding site for translation initiation and a transcription terminator.Large numbers of suitable vectors and promoters/enhancers, will be knownto those of skill in the art, but any plasmid or vector,promoter/enhancer may be used as long as it is replicable and functionalin the host. The vector may also include appropriate sequences forselection and/or amplification of expression. For this the vector willcomprise one or more phenotypic selectable/amplifiable markers. Suchmarkers are also well known to those skilled in the art.

[0062] In a further aspect, the present invention provides host cellscomprising a vector of the invention, and capable of expressing anucleotide sequence of the invention. The host cells can be, forexample, a higher eukaryotic cell, such as a mammalian cell or a lowereukaryotic cell, such as a yeast cell or a prokaryotic cell such as abacterial cell. Suitable prokaryotic hosts for transformation includeE-coli. Suitable eukaryotic hosts include insect cells, e.g. SF9 cells.Tni cells, Hi5 cells, and mammalian cells, e.g. HEK HeLa, COS, CHO, NSO3T3 (fibroblast cell line). Cells expressing a nucleotide sequence ofthe invention can be lysed to obtain the polypeptide of the invention,which may optionally be purified.

[0063] Cell free translation systems can also be employed to producesuch proteins using RNAs derived from the DNA constructs of the presentinvention.

[0064] A further aspect of the invention is a method of producing apolypeptide of the invention, which method comprises introducing into anappropriate cell line a vector comprising a polynucleotide as definedherein under conditions suitable for obtaining expression of thepolypeptide.

[0065] Methods of Screening

[0066] The present invention further provides a method foridentification of a compound which modulates Tec kinase activity.Compounds which modulate Tec kinase activity are those which inhibit orenhance the function of the Tec kinase polypeptides (e.g. to act asantagonists or agonists of the Tec kinase protein function). In generalterms, the screen for such compounds will comprise contacting apolypeptide of the invention with a test compound, and then detectingany enhancement or inhibition of polypeptide activity that results(compared to the activity that would occur in the absence of the testcompound). By contacting, it is meant that the test compound and thepolypeptide of the invention are in such proximity that they are able tointeract biologically. The polypeptides of the invention may be used inhigh throughput screens, thus enabling large numbers of compounds to bestudied.

[0067] The activity that is detected is preferably phospho-transferactivity. Phospho-transfer involves the transfer of a phosphate from ATPto a tyrosine residue contained within the substrate. Phospho-transferactivity may be detected by the inclusion of a target polypeptide in theassay. More particularly, polypeptides of the invention may be used inany suitable tyrosine kinase assay, for example, by a time-resolvedfluorescence assay, such as homogenous time-resolved fluorescence (HTRF, Packard Instrument Company) Kolb et al., DDT Vol. 3, 333-342; in-platebinding assays including calorimetric and luminescent read-outs andtime-resolved fluorescence, see for example Farley et al., Anal Biochem(1992) 203, 151-157, Lehel et al., (1997) Anal. Biochem. 244, 340-346and Braunwalder et al., (1996) Anal. Biochem. 238, 340-346; orradiometric assays, for example, using ³³P or ³²P, such as scintillationproximity assay (SPA, Amersham International). Examples of time-resolvedfluorescence assays are: in-plate time-resolved fluorescence (trf) assay(Delfia®, Wallac Oy). Preferably a time-resolved fluorescence assay isused, most preferably homogenous time-resolved fluorescence.

[0068] In a preferred aspect, the method of screening is carried outusing cells expressing a polypeptide of the invention, and incubatingsuch cells with the test compound, optionally in the presence of a Teckinase ligand.

[0069] Methods of Treatment

[0070] A further aspect of the present invention is a compound whichmodulates Tec kinase activity and is identifiable by screeningtechniques referred to above. Preferably, the compound has beenidentified using the above screening techniques. The compounds may beagonists or antagonists of the Tec kinase polypeptide, but preferablyare antagonists. The compounds include, for example, aptamers,polypeptides, antibodies and small molecules. The compounds may beuseful in the treatment and/or prophylaxis of disorders that areresponsive to modulation of Tec kinase activity. For example, it hasbeen demonstrated in vivo that Itk-deficient mice are unable toestablish TH2 cells suggesting that Itk has a role in mediating thedevelopment of IL-4 producing TH2 cells (Fowell et al. (1999) Immunity11:399-409). The absence of this cytokine in Itk-deficient mice suggeststhat compounds which modulate Tec kinases may be useful in the treatmentof inflammatory diseases such as, for example, asthma.

[0071] One particular aspect of the invention is the use of a compoundwhich modulates Tec kinase activity and is identifiable by the screeningtechniques of the invention, for the manufacture of a medicament for thetreatment or prophylaxis of disorders that are responsive to modulationof the activity of Tec kinase activity, such as inflammation.

[0072] Another aspect of the invention is a method of treating a subjecthaving a disorder which is responsive to modulation of Tec kinaseactivity, such as inflammation, which method comprises administering toa subject an effective amount of a compound identifiable by thescreening techniques of the invention.

[0073] Examples of inflammatory conditions include: asthma, allergicrhinitis, URID (upper respiratory inflammatory disease), adultrespiratory distress syndrome; arthritic conditions such as rheumatoidarthritis, rheumatoid spondylitis, and osteoarthritis; inflammatory eyeconditions such as uveitis (including iritis) and conjunctivitis;inflammatory bowel conditions such as Crohn's disease, ulcerativecolitis and distal proctitis; periodontal disease; esophagitis,inflammatory skin conditions such as psoriasis, eczema and dermatitis.Preferred inflammatory conditions are asthma, allergic rhinitis and URID(upper respiratory inflammatory disease).

[0074] Where the Tec kinase is Btk, preferably the disorder is selectedfrom inflammatory conditions (described above); diseases with a B cellcomponent such as B cell leukaemias and SLE (Systemic Lupuserythematosis); and diseases associated with platelet function.

[0075] Substances identified according to the screening methods outlinedabove may be formulated with standard pharmaceutically acceptablecarriers and/or excipients as is routine in the pharmaceutical art. Theexact nature of a formulation will depend upon several factors includingthe particular substance to be administered and the desired route ofadministration. The substances may be administered by enteral orparenteral routes such as via oral, buccal, anal, pulmonary,intravenous, intra-arterial, intramuscular, intraperitoneal, topical orother appropriate administration routes.

[0076] Therapeutically effective amounts of such compounds can bereadily determined by those skilled in the art using, e.g. dose-responsestudies. The dose of agent may be determined according to variousparameters, especially according to the substance used; the age, weightand condition of the patient to be treated; the route of administrationand the required regime. A suitable dose may however be from 0.01 to 50mg/kg body weight such as 1 to 40 mg/kg body weight. A physician will beable to determine the required route of administration and dosage forany particular patient.

BRIEF DESCRIPTION OF THE FIGURES:

[0077]FIG. 1 shows the oligonucleotide sequences used to generate thetruncated Itk construct shown in FIG. 2.

[0078]FIG. 2 shows the polynucleotide sequence of the truncated Itkconstruct (including cloning sites, underlined).

[0079]FIG. 3 shows the polynucleotide and translated polypeptidesequence of the truncated ltk construct.

[0080]FIG. 4 shows an alignment of the polypeptide sequences of Itk (asin Swissprot data base accession number: Q08881) and the truncatedconstruct showing PH; SH3; SH2 and kinase domains. The domain boundariesare as defined by Swissprot entry Q08881: PH 4-111; SH3 171-23; SH2239-338; Kinase 363-615. Note: tITK=truncated ITK.

[0081]FIG. 5 shows an alignment of the polypeptide sequences of Tecfamily kinases (SwissProt database):

[0082] Btk (human)—Accession no. Q06187, Btk (mouse)—Accession no.P35991, Itk (human)—Accession no. 008881, Itk (mouse)—Accession no.Q03526, Tec (human)—Accession no. P42680, Tec (mouse)—Accession no.P24604, Bmx (human)—Accession no. P51813, Txk (human)—P42681/Q14220, andTxk (mouse)—Accession no. P42682, showing the PH domain (Bold, italicand boxed), Tec homology domain (Italic and boxed), SH3 domain (Bold andboxed) and SH2 domain (Boxed).

[0083]FIG. 6 shows a schematic representation of the domain structure ofTec kinases.

[0084]FIG. 7 shows the oligonucleotide sequences used to generate thetruncated Btk construct shown in FIG. 9.

[0085]FIG. 8 shows the translated polypeptide sequence of the truncatedBtk construct.

[0086]FIG. 9 shows the polynucleotide sequence of the truncated Btkconstruct. Note: the full length sequence is available on the genembldatabase accession 20 number X58957.

EXAMPLES Example 1A

[0087] Generation of the Truncated Itk Construct

[0088] To generate an active form of Itk oligonucleotide, PCR primerswere designed to amplify, from cDNA, a single region of Itkcorresponding to the combined SH3, SH2 and kinase domains and toincorporate a start methionine and restriction endonuclease sites forcloning the construct. The Oligonucleotide sequences used to generatethe truncated Itk construct are shown in FIG. 1. A T cell cDNA librarywas used as a source of template DNA (generation of library described inBiotechniques (1998) 25:85-92). A PCR product was cloned (FIG. 2).sequenced and used to generate a recombinant baculovirus for infectionof SF9 insect cells using standard molecular biological techniques (seefor example, Sambrook et al, Molecular Cloning: a Laboratory Manual, 2_(nd) Edition, CSH Laboratory Press, 1989).

Example 1 B

[0089] Protein Expression and Assay Formation

[0090] Insect cell pellets infected with the recombinant baculovirusdescribed in Example 1A were homogenised in 40 mM HEPES (pH 7.4), 100 mMNaCl 2 mM EDTA, 10% glycerol, 0.1 mM vanadate and protease inhibitors.The 100 000 g supernatant was stored at −85° C. Stored lysates werethawed on ice, ATP and MgCl₂ (0.1 mM and 10 mM) were added. Followingincubation on ice the kinase was diluted in 40 mM HEPES (pH 7.4). Thekinase reaction mixture contained 40 mM HEPES (pH 7.4). 10 mM MgCl₂,0.05 mM ATP, 0.0005 mM peptide (Biotin-AAAEEIYGEI). The reaction wasstopped by the addition of EDTA (25 mM). The amount of phosphopeptidewas quantitated by homogeneous time resolved fluorescence as describedin Kolb et al. (1998) Drug Discovery Today 3:333-342. An increase in thelevel of fluorescence indicates an increase in kinase activity.

[0091] The Km for ATP and peptide was determined to be 0.039+/−0.011mMand 0.480+/−0.183 uM respectively.

Example 1 C Screening Compounds for Modulation of Itk Activity

[0092] The table below shows inhibitors of Itk activity identified usingthe screen described in Example 1B above.

[0093] Compound Testing in Kinase Assay

[0094] Order of additions:

[0095] Compound to well

[0096] Enzyme to well

[0097] 15 min pre-incubation

[0098] Substrates (ATP, src-peptide) added

[0099] 30 min incubation

[0100] Reaction stopped with EDTA

[0101] HTRF reagents (APC, Eu labelled antibody (antiPY antibody)) added

[0102] Stand for 20 mins

[0103] Read signal on plate reader UK:ITK Average Average Class NamePIC50 IC50 (μM)

flavone 6.09 0.813

flavone 6.09 0.813

flavone 5.78 1.660

Example 1 D

[0104] Performance of Itk Screen

[0105] The following data demonstrate the robustness of the Itk assay.Repetition of the assay described above over a period between 12, Aug.1999 and 24, Feb. 2000 (Batch 1) and 22, May 2000 and 25, May 2000(Batch 2) demonstrates: (i) it is possible to generate large amounts oftruncated enzyme which are stable over a long time, and (ii) the assaygives a high frequency of comparable results upon repeat testing. Batch1 Batch 2 Date Aug. 12, Feb. 24, 2000 Mar. 22, 2000 May 25, 2000 1999Control 7076 ± 735 6373 ± 461 6978 ± 341 5616 ± 66 Blank  175 ± 7  193 ±31  140 ± 15  120 ± 3 Signal/  40  33  50  47 Noise

Example 2A

[0106] Generation of the Truncated Btk Construct

[0107] To generate an active form of Btk oligonucleotide, PCR primerswere designed to amplify, from cDNA, a single region of Btkcorresponding to the combined SH3, SH2 and kinase domains and toincorporate a start methionine and restriction endonuclease sites forcloning the construct. The Oligonucleotide sequences used to generatethe truncated Btk construct are shown in FIG. 7. A PCR product wascloned, sequenced and used to generate a recombinant baculovirus forinfection of SF9 insect cells using standard molecular biologicaltechniques (see for example, Sambrook et al, Molecular Cloning: aLaboratory Manual, 2^(nd) Edition, CSH Laboratory Press, 1989).

Example 2B

[0108] Protein Expression and Assay Formation

[0109] Insect cell pellets infected with the recombinant baculovirusdescribed in Example 2A were homogenised in 40 mM HEPES (pH 7.4), 100 mMNaCl 2 mM EDTA, 10% glycerol, 0.1 mM vanadate and protease inhibitors.The 100 000 g supernatant was stored at −85° C. Stored lysates werethawed on ice and diluted in 40 mM HEPES (pH 7.4). The kinase reactionmixture contained 40mM HEPES pH7.4; 80 mM MgCl₂; 1300 μM ATP; 500 nMpeptide (Biotin-AAAEEIYGEI-NH2). The reaction was stopped by theaddition of EDTA (0.16 M). The amount of phosphopeptide was quantitatedby homogeneous time resolved fluorescence as described in Kolb et al.(1998) Drug Discovery Today 3:333-342. An increase in the level offluorescence indicates an increase in kinase activity.

[0110] The Km for ATP was determined to be 1.69+/−0.36 mM

Example 2C

[0111] Screening Compounds for Modulation of Btk Activity

[0112] The table below shows an inhibitor of Btk activity identifiedusing the screen described in Example 2B above.

[0113] Compound Testing in Kinase Assay

[0114] Order of additions:

[0115] Compound to well

[0116] Enzyme to well

[0117] 15 min pre-incubation

[0118] Substrates (ATP, src-peptide) added

[0119] 30 min incubation

[0120] Reaction stopped with EDTA

[0121] HTRF reagents (APC. Eu labelled antibody (antiPY antibody)) added

[0122] Stand for 20 mins

[0123] Read signal on plate reader

[0124] Compound: Staurosporin

[0125] pIC₅₀=6.83

[0126] Note: PIC₅₀ is −log10 IC₅₀in molar, a higher PIC₅₀ indicatesgreater potency.

1 23 1 9 PRT Artificial Sequence SITE (1)..(2) Xaa is any one amino acidresidue 1 Xaa Xaa Val Xaa Ala Xaa Tyr Asp Xaa 1 5 2 7 PRT ArtificialSequence SITE (1) Xaa is Ile or Leu 2 Xaa Xaa Xaa Tyr Glu Trp Tyr 1 5 37 PRT Artificial Sequence SITE (2) Xaa is Leu or Phe 3 Gly Xaa Xaa TyrXaa Xaa Trp 1 5 4 31 DNA Artificial Sequence Description of ArtificialSequence 5 prime oligo 4 gatcggatcc atgcctgaag aaactgtggt c 31 5 28 DNAArtificial Sequence Description of Artificial Sequence 3 prime oligo 5gatcggatcc ctaaagtcct gattctgc 28 6 1368 DNA Artificial Sequence CDS(7)..(1362) Description of Artificial Sequence Truncated Itk construct 6ggatcc atg cct gaa gaa act gtg gtc att gcc tta tat gac tac caa 48 MetPro Glu Glu Thr Val Val Ile Ala Leu Tyr Asp Tyr Gln 1 5 10 acc aat gatcct cag gaa ctc gca ctg cgg cgc aac gaa gag tac tgc 96 Thr Asn Asp ProGln Glu Leu Ala Leu Arg Arg Asn Glu Glu Tyr Cys 15 20 25 30 ctg ctg gacagt tct gag att cac tgg tgg aga gtc cag gac agg aat 144 Leu Leu Asp SerSer Glu Ile His Trp Trp Arg Val Gln Asp Arg Asn 35 40 45 ggg cat gaa ggatat gta cca agc agt tat ctg gtg gaa aaa tct cca 192 Gly His Glu Gly TyrVal Pro Ser Ser Tyr Leu Val Glu Lys Ser Pro 50 55 60 aat aat ctg gaa acctat gag tgg tac aat aag agt atc agc cga gac 240 Asn Asn Leu Glu Thr TyrGlu Trp Tyr Asn Lys Ser Ile Ser Arg Asp 65 70 75 aaa gct gaa aaa ctt cttttg gac aca ggc aaa gaa gga gcc ttc atg 288 Lys Ala Glu Lys Leu Leu LeuAsp Thr Gly Lys Glu Gly Ala Phe Met 80 85 90 gta agg gat tcc agg act gcagga aca tac acc gtg tct gtt ttc acc 336 Val Arg Asp Ser Arg Thr Ala GlyThr Tyr Thr Val Ser Val Phe Thr 95 100 105 110 aag gct gtt gta agt gagaac aat ccc tgt ata aag cat tat cac atc 384 Lys Ala Val Val Ser Glu AsnAsn Pro Cys Ile Lys His Tyr His Ile 115 120 125 aag gaa aca aat gac aatcct aag cga tac tat gtg gct gaa aag tat 432 Lys Glu Thr Asn Asp Asn ProLys Arg Tyr Tyr Val Ala Glu Lys Tyr 130 135 140 gtg ttc gat tcc atc cctctt ctc atc aac tat cac caa cat aat gga 480 Val Phe Asp Ser Ile Pro LeuLeu Ile Asn Tyr His Gln His Asn Gly 145 150 155 gga ggc ctg gtg act cgactc cgg tat cca gtt tgt ttt ggg agg cag 528 Gly Gly Leu Val Thr Arg LeuArg Tyr Pro Val Cys Phe Gly Arg Gln 160 165 170 aaa gcc cca gtt aca gcaggg ctg aga tac ggg aaa tgg gtg atc gac 576 Lys Ala Pro Val Thr Ala GlyLeu Arg Tyr Gly Lys Trp Val Ile Asp 175 180 185 190 ccc tca gag ctc actttt gtg caa gag att ggc agt ggg caa ttt ggg 624 Pro Ser Glu Leu Thr PheVal Gln Glu Ile Gly Ser Gly Gln Phe Gly 195 200 205 ttg gtg cat ctg ggctac tgg ctc aac aag gac aag gtg gct atc aaa 672 Leu Val His Leu Gly TyrTrp Leu Asn Lys Asp Lys Val Ala Ile Lys 210 215 220 acc att cgg gaa ggggct atg tca gaa gag gac ttc ata gag gag gct 720 Thr Ile Arg Glu Gly AlaMet Ser Glu Glu Asp Phe Ile Glu Glu Ala 225 230 235 gaa gta atg atg aaactc tct cat ccc aaa ctg gtg cag ctg tat ggg 768 Glu Val Met Met Lys LeuSer His Pro Lys Leu Val Gln Leu Tyr Gly 240 245 250 gtg tgc ctg gag caggcc ccc atc tgc ctg gtg ttt gag ttc atg gag 816 Val Cys Leu Glu Gln AlaPro Ile Cys Leu Val Phe Glu Phe Met Glu 255 260 265 270 cac ggc tgc ctgtca gat tat cta cgc acc cag cgg gga ctt ttt gct 864 His Gly Cys Leu SerAsp Tyr Leu Arg Thr Gln Arg Gly Leu Phe Ala 275 280 285 gca gag acc ctgctg ggc atg tgt ctg gat gtg tgt gag ggc atg gcc 912 Ala Glu Thr Leu LeuGly Met Cys Leu Asp Val Cys Glu Gly Met Ala 290 295 300 tac ctg gaa gaggca tgt gtc atc cac aga gac ttg gct gcc aga aat 960 Tyr Leu Glu Glu AlaCys Val Ile His Arg Asp Leu Ala Ala Arg Asn 305 310 315 tgt ttg gtg ggagaa aac caa gtc atc aag gtg tct gac ttt ggg atg 1008 Cys Leu Val Gly GluAsn Gln Val Ile Lys Val Ser Asp Phe Gly Met 320 325 330 aca agg ttc gttctg gat gat cag tac acc agt tcc aca ggc acc aaa 1056 Thr Arg Phe Val LeuAsp Asp Gln Tyr Thr Ser Ser Thr Gly Thr Lys 335 340 345 350 ttc ccg gtgaag tgg gca tcc cca gag gtt ttc tct ttc agt cgc tat 1104 Phe Pro Val LysTrp Ala Ser Pro Glu Val Phe Ser Phe Ser Arg Tyr 355 360 365 agc agc aagtcc gat gtg tgg tca ttt ggt gtg ctg atg tgg gaa gtt 1152 Ser Ser Lys SerAsp Val Trp Ser Phe Gly Val Leu Met Trp Glu Val 370 375 380 ttc agt gaaggc aaa atc ccg tat gaa aac cga agc aac tca gag gtg 1200 Phe Ser Glu GlyLys Ile Pro Tyr Glu Asn Arg Ser Asn Ser Glu Val 385 390 395 gtg gaa gacatc agt acc gga ttt cgg ttg tac aag ccc cgg ctg gcc 1248 Val Glu Asp IleSer Thr Gly Phe Arg Leu Tyr Lys Pro Arg Leu Ala 400 405 410 tcc aca cacgtc tac cag att atg aat cac tgc tgg aaa gag aga cca 1296 Ser Thr His ValTyr Gln Ile Met Asn His Cys Trp Lys Glu Arg Pro 415 420 425 430 gaa gatcgg cca gcc ttc tcc aga ctg ctg cgt caa ctg gct gaa att 1344 Glu Asp ArgPro Ala Phe Ser Arg Leu Leu Arg Gln Leu Ala Glu Ile 435 440 445 gca gaatca gga ctt tag ggatcc 1368 Ala Glu Ser Gly Leu 450 7 451 PRT ArtificialSequence Description of Artificial Sequence Truncated Itk construct 7Met Pro Glu Glu Thr Val Val Ile Ala Leu Tyr Asp Tyr Gln Thr Asn 1 5 1015 Asp Pro Gln Glu Leu Ala Leu Arg Arg Asn Glu Glu Tyr Cys Leu Leu 20 2530 Asp Ser Ser Glu Ile His Trp Trp Arg Val Gln Asp Arg Asn Gly His 35 4045 Glu Gly Tyr Val Pro Ser Ser Tyr Leu Val Glu Lys Ser Pro Asn Asn 50 5560 Leu Glu Thr Tyr Glu Trp Tyr Asn Lys Ser Ile Ser Arg Asp Lys Ala 65 7075 80 Glu Lys Leu Leu Leu Asp Thr Gly Lys Glu Gly Ala Phe Met Val Arg 8590 95 Asp Ser Arg Thr Ala Gly Thr Tyr Thr Val Ser Val Phe Thr Lys Ala100 105 110 Val Val Ser Glu Asn Asn Pro Cys Ile Lys His Tyr His Ile LysGlu 115 120 125 Thr Asn Asp Asn Pro Lys Arg Tyr Tyr Val Ala Glu Lys TyrVal Phe 130 135 140 Asp Ser Ile Pro Leu Leu Ile Asn Tyr His Gln His AsnGly Gly Gly 145 150 155 160 Leu Val Thr Arg Leu Arg Tyr Pro Val Cys PheGly Arg Gln Lys Ala 165 170 175 Pro Val Thr Ala Gly Leu Arg Tyr Gly LysTrp Val Ile Asp Pro Ser 180 185 190 Glu Leu Thr Phe Val Gln Glu Ile GlySer Gly Gln Phe Gly Leu Val 195 200 205 His Leu Gly Tyr Trp Leu Asn LysAsp Lys Val Ala Ile Lys Thr Ile 210 215 220 Arg Glu Gly Ala Met Ser GluGlu Asp Phe Ile Glu Glu Ala Glu Val 225 230 235 240 Met Met Lys Leu SerHis Pro Lys Leu Val Gln Leu Tyr Gly Val Cys 245 250 255 Leu Glu Gln AlaPro Ile Cys Leu Val Phe Glu Phe Met Glu His Gly 260 265 270 Cys Leu SerAsp Tyr Leu Arg Thr Gln Arg Gly Leu Phe Ala Ala Glu 275 280 285 Thr LeuLeu Gly Met Cys Leu Asp Val Cys Glu Gly Met Ala Tyr Leu 290 295 300 GluGlu Ala Cys Val Ile His Arg Asp Leu Ala Ala Arg Asn Cys Leu 305 310 315320 Val Gly Glu Asn Gln Val Ile Lys Val Ser Asp Phe Gly Met Thr Arg 325330 335 Phe Val Leu Asp Asp Gln Tyr Thr Ser Ser Thr Gly Thr Lys Phe Pro340 345 350 Val Lys Trp Ala Ser Pro Glu Val Phe Ser Phe Ser Arg Tyr SerSer 355 360 365 Lys Ser Asp Val Trp Ser Phe Gly Val Leu Met Trp Glu ValPhe Ser 370 375 380 Glu Gly Lys Ile Pro Tyr Glu Asn Arg Ser Asn Ser GluVal Val Glu 385 390 395 400 Asp Ile Ser Thr Gly Phe Arg Leu Tyr Lys ProArg Leu Ala Ser Thr 405 410 415 His Val Tyr Gln Ile Met Asn His Cys TrpLys Glu Arg Pro Glu Asp 420 425 430 Arg Pro Ala Phe Ser Arg Leu Leu ArgGln Leu Ala Glu Ile Ala Glu 435 440 445 Ser Gly Leu 450 8 620 PRT Homosapiens 8 Met Asn Asn Phe Ile Leu Leu Glu Glu Gln Leu Ile Lys Lys SerGln 1 5 10 15 Gln Lys Arg Arg Thr Ser Pro Ser Asn Phe Lys Val Arg PhePhe Val 20 25 30 Leu Thr Lys Ala Ser Leu Ala Tyr Phe Glu Asp Arg His GlyLys Lys 35 40 45 Arg Thr Leu Lys Gly Ser Ile Glu Leu Ser Arg Ile Lys CysVal Glu 50 55 60 Ile Val Lys Ser Asp Ile Ser Ile Pro Cys His Tyr Lys TyrPro Phe 65 70 75 80 Gln Val Val His Asp Asn Tyr Leu Leu Tyr Val Phe AlaPro Asp Arg 85 90 95 Glu Ser Arg Gln Arg Trp Val Leu Ala Leu Lys Glu GluThr Arg Asn 100 105 110 Asn Asn Ser Leu Val Pro Lys Tyr His Pro Asn PheTrp Met Asp Gly 115 120 125 Lys Trp Arg Cys Cys Ser Gln Leu Glu Lys LeuAla Thr Gly Cys Ala 130 135 140 Gln Tyr Asp Pro Thr Lys Asn Ala Ser LysLys Pro Leu Pro Pro Thr 145 150 155 160 Pro Glu Asp Asn Arg Arg Pro LeuTrp Glu Pro Glu Glu Thr Val Val 165 170 175 Ile Ala Leu Tyr Asp Tyr GlnThr Asn Asp Pro Gln Glu Leu Ala Leu 180 185 190 Arg Arg Asn Glu Glu TyrCys Leu Leu Asp Ser Ser Glu Ile His Trp 195 200 205 Trp Arg Val Gln AspArg Asn Gly His Glu Gly Tyr Val Pro Ser Ser 210 215 220 Tyr Leu Val GluLys Ser Pro Asn Asn Leu Glu Thr Tyr Glu Trp Tyr 225 230 235 240 Asn LysSer Ile Ser Arg Asp Lys Ala Glu Lys Leu Leu Leu Asp Thr 245 250 255 GlyLys Glu Gly Ala Phe Met Val Arg Asp Ser Arg Thr Ala Gly Thr 260 265 270Tyr Thr Val Ser Val Phe Thr Lys Ala Val Val Ser Glu Asn Asn Pro 275 280285 Cys Ile Lys His Tyr His Ile Lys Glu Thr Asn Asp Asn Pro Lys Arg 290295 300 Tyr Tyr Val Ala Glu Lys Tyr Val Phe Asp Ser Ile Pro Leu Leu Ile305 310 315 320 Asn Tyr His Gln His Asn Gly Gly Gly Leu Val Thr Arg LeuArg Tyr 325 330 335 Pro Val Cys Phe Gly Arg Gln Lys Ala Pro Val Thr AlaGly Leu Arg 340 345 350 Tyr Gly Lys Trp Val Ile Asp Pro Ser Glu Leu ThrPhe Val Gln Glu 355 360 365 Ile Gly Ser Gly Gln Phe Gly Leu Val His LeuGly Tyr Trp Leu Asn 370 375 380 Lys Asp Lys Val Ala Ile Lys Thr Ile ArgGlu Gly Ala Met Ser Glu 385 390 395 400 Glu Asp Phe Ile Glu Glu Ala GluVal Met Met Lys Leu Ser His Pro 405 410 415 Lys Leu Val Gln Leu Tyr GlyVal Cys Leu Glu Gln Ala Pro Ile Cys 420 425 430 Leu Val Phe Glu Phe MetGlu His Gly Cys Leu Ser Asp Tyr Leu Arg 435 440 445 Thr Gln Arg Gly LeuPhe Ala Ala Glu Thr Leu Leu Gly Met Cys Leu 450 455 460 Asp Val Cys GluGly Met Ala Tyr Leu Glu Glu Ala Cys Val Ile His 465 470 475 480 Arg AspLeu Ala Ala Arg Asn Cys Leu Val Gly Glu Asn Gln Val Ile 485 490 495 LysVal Ser Asp Phe Gly Met Thr Arg Phe Val Leu Asp Asp Gln Tyr 500 505 510Thr Ser Ser Thr Gly Thr Lys Phe Pro Val Lys Trp Ala Ser Pro Glu 515 520525 Val Phe Ser Phe Ser Arg Tyr Ser Ser Lys Ser Asp Val Trp Ser Phe 530535 540 Gly Val Leu Met Trp Glu Val Phe Ser Glu Gly Lys Ile Pro Tyr Glu545 550 555 560 Asn Arg Ser Asn Ser Glu Val Val Glu Asp Ile Ser Thr GlyPhe Arg 565 570 575 Leu Tyr Lys Pro Arg Leu Ala Ser Thr His Val Tyr GlnIle Met Asn 580 585 590 His Cys Trp Lys Glu Arg Pro Glu Asp Arg Pro AlaPhe Ser Arg Leu 595 600 605 Leu Arg Gln Leu Ala Glu Ile Ala Glu Ser GlyLeu 610 615 620 9 395 PRT Homo sapiens 9 Met Ala Ala Val Ile Leu Glu SerIle Phe Leu Lys Arg Ser Gln Gln 1 5 10 15 Lys Lys Lys Thr Ser Pro LeuAsn Phe Lys Lys Arg Leu Phe Leu Leu 20 25 30 Thr Val His Lys Leu Ser TyrTyr Glu Tyr Asp Phe Glu Arg Gly Arg 35 40 45 Arg Gly Ser Lys Lys Gly SerIle Asp Val Glu Lys Ile Thr Cys Val 50 55 60 Glu Thr Val Val Pro Glu LysAsn Pro Pro Pro Glu Arg Gln Ile Pro 65 70 75 80 Arg Arg Gly Glu Glu SerSer Glu Met Glu Gln Ile Ser Ile Ile Glu 85 90 95 Arg Phe Pro Tyr Pro PheGln Val Val Tyr Asp Glu Gly Pro Leu Tyr 100 105 110 Val Phe Ser Pro ThrGlu Glu Leu Arg Lys Arg Trp Ile His Gln Leu 115 120 125 Lys Asn Val IleArg Tyr Asn Ser Asp Leu Val Gln Lys Tyr His Pro 130 135 140 Cys Phe TrpIle Asp Gly Gln Tyr Leu Cys Cys Ser Gln Thr Ala Lys 145 150 155 160 AsnAla Met Gly Cys Gln Ile Leu Glu Asn Arg Asn Gly Ser Leu Lys 165 170 175Pro Gly Ser Ser His Arg Lys Thr Lys Lys Pro Leu Pro Pro Thr Pro 180 185190 Glu Glu Asp Gln Ile Leu Lys Lys Pro Leu Pro Pro Glu Pro Ala Ala 195200 205 Ala Pro Val Ser Thr Ser Glu Leu Lys Lys Val Val Ala Leu Tyr Asp210 215 220 Tyr Met Pro Met Asn Ala Asn Asp Leu Gln Leu Arg Lys Gly AspGlu 225 230 235 240 Tyr Phe Ile Leu Glu Glu Ser Asn Leu Pro Trp Trp ArgAla Arg Asp 245 250 255 Lys Asn Gly Gln Glu Gly Tyr Ile Pro Ser Asn TyrVal Thr Glu Ala 260 265 270 Glu Asp Ser Ile Glu Met Tyr Glu Trp Tyr SerLys His Met Thr Arg 275 280 285 Ser Gln Ala Glu Gln Leu Leu Lys Gln GluGly Lys Glu Gly Gly Phe 290 295 300 Ile Val Arg Asp Ser Ser Lys Ala GlyLys Tyr Thr Val Ser Val Phe 305 310 315 320 Ala Lys Ser Thr Gly Asp ProGln Gly Val Ile Arg His Tyr Val Val 325 330 335 Cys Ser Thr Pro Gln SerGln Tyr Tyr Leu Ala Glu Lys His Leu Phe 340 345 350 Ser Thr Ile Pro GluLeu Ile Asn Tyr His Gln His Asn Ser Ala Gly 355 360 365 Leu Ile Ser ArgLeu Lys Tyr Pro Val Ser Gln Gln Asn Lys Asn Ala 370 375 380 Pro Ser ThrAla Gly Leu Gly Tyr Gly Ser Trp 385 390 395 10 395 PRT Mus musculus 10Met Ala Ala Val Ile Leu Glu Ser Ile Phe Leu Lys Arg Ser Gln Gln 1 5 1015 Lys Lys Lys Thr Ser Pro Leu Asn Phe Lys Lys Arg Leu Phe Leu Leu 20 2530 Thr Val His Lys Leu Ser Tyr Tyr Glu Tyr Asp Phe Glu Arg Gly Arg 35 4045 Arg Gly Ser Lys Lys Gly Ser Ile Asp Val Glu Lys Ile Thr Cys Val 50 5560 Glu Thr Val Ile Pro Glu Lys Asn Pro Pro Pro Glu Arg Gln Ile Pro 65 7075 80 Arg Arg Gly Glu Glu Ser Ser Glu Met Glu Gln Ile Ser Ile Ile Glu 8590 95 Arg Phe Pro Tyr Pro Phe Gln Val Val Tyr Asp Glu Gly Pro Leu Tyr100 105 110 Val Phe Ser Pro Thr Glu Glu Leu Arg Lys Arg Trp Ile His GlnLeu 115 120 125 Lys Asn Val Ile Arg Tyr Asn Ser Asp Leu Val Gln Lys TyrHis Pro 130 135 140 Cys Phe Trp Ile Asp Gly Gln Tyr Leu Cys Cys Ser GlnThr Ala Lys 145 150 155 160 Asn Ala Met Gly Cys Gln Ile Leu Glu Asn ArgAsn Gly Ser Leu Lys 165 170 175 Pro Gly Ser Ser His Arg Lys Thr Lys LysPro Leu Pro Pro Thr Pro 180 185 190 Glu Glu Asp Gln Ile Leu Lys Lys ProLeu Pro Pro Glu Pro Thr Ala 195 200 205 Ala Pro Ile Ser Thr Thr Glu LeuLys Lys Val Val Ala Leu Tyr Asp 210 215 220 Tyr Met Pro Met Asn Ala AsnAsp Leu Gln Leu Arg Lys Gly Glu Glu 225 230 235 240 Tyr Phe Ile Leu GluGlu Ser Asn Leu Pro Trp Trp Arg Ala Arg Asp 245 250 255 Lys Asn Gly GlnGlu Gly Tyr Ile Pro Ser Asn Tyr Ile Thr Glu Ala 260 265 270 Glu Asp SerIle Glu Met Tyr Glu Trp Tyr Ser Lys His Met Thr Arg 275 280 285 Ser GlnAla Glu Gln Leu Leu Lys Gln Glu Gly Lys Glu Gly Gly Phe 290 295 300 IleVal Arg Asp Ser Ser Lys Ala Gly Lys Tyr Thr Val Ser Val Phe 305 310 315320 Ala Lys Ser Thr Gly Glu Pro Gln Gly Val Ile Arg His Tyr Val Val 325330 335 Cys Ser Thr Pro Gln Ser Gln Tyr Tyr Leu Ala Glu Lys His Leu Phe340 345 350 Ser Thr Ile Pro Glu Leu Ile Asn Tyr His Gln His Asn Ser AlaGly 355 360 365 Leu Ile Ser Arg Leu Lys Tyr Pro Val Ser Lys Gln Asn LysAsn Ala 370 375 380 Pro Ser Thr Ala Gly Leu Gly Tyr Gly Ser Trp 385 390395 11 356 PRT Homo sapiens 11 Met Asn Asn Phe Ile Leu Leu Glu Glu GlnLeu Ile Lys Lys Ser Gln 1 5 10 15 Gln Lys Arg Arg Thr Ser Pro Ser AsnPhe Lys Val Arg Phe Phe Val 20 25 30 Leu Thr Lys Ala Ser Leu Ala Tyr PheGlu Asp Arg His Gly Lys Lys 35 40 45 Arg Thr Leu Lys Gly Ser Ile Glu LeuSer Arg Ile Lys Cys Val Glu 50 55 60 Ile Val Lys Ser Asp Ile Ser Ile ProCys His Tyr Lys Tyr Pro Phe 65 70 75 80 Gln Val Val His Asp Asn Tyr LeuLeu Tyr Val Phe Ala Pro Asp Arg 85 90 95 Glu Ser Arg Gln Arg Trp Val LeuAla Leu Lys Glu Glu Thr Arg Asn 100 105 110 Asn Asn Ser Leu Val Pro LysTyr His Pro Asn Phe Trp Met Asp Gly 115 120 125 Lys Trp Arg Cys Cys SerGln Leu Glu Lys Leu Ala Thr Gly Cys Ala 130 135 140 Gln Tyr Asp Pro ThrLys Asn Ala Ser Lys Lys Pro Leu Pro Pro Thr 145 150 155 160 Pro Glu AspAsn Arg Arg Pro Leu Trp Glu Pro Glu Glu Thr Val Val 165 170 175 Ile AlaLeu Tyr Asp Tyr Gln Thr Asn Asp Pro Gln Glu Leu Ala Leu 180 185 190 ArgArg Asn Glu Glu Tyr Cys Leu Leu Asp Ser Ser Glu Ile His Trp 195 200 205Trp Arg Val Gln Asp Arg Asn Gly His Glu Gly Tyr Val Pro Ser Ser 210 215220 Tyr Leu Val Glu Lys Ser Pro Asn Asn Leu Glu Thr Tyr Glu Trp Tyr 225230 235 240 Asn Lys Ser Ile Ser Arg Asp Lys Ala Glu Lys Leu Leu Leu AspThr 245 250 255 Gly Lys Glu Gly Ala Phe Met Val Arg Asp Ser Arg Thr AlaGly Thr 260 265 270 Tyr Thr Val Ser Val Phe Thr Lys Ala Val Val Ser GluAsn Asn Pro 275 280 285 Cys Ile Lys His Tyr His Ile Lys Glu Thr Asn AspAsn Pro Lys Arg 290 295 300 Tyr Tyr Val Ala Glu Lys Tyr Val Phe Asp SerIle Pro Leu Leu Ile 305 310 315 320 Asn Tyr His Gln His Asn Gly Gly GlyLeu Val Thr Arg Leu Arg Tyr 325 330 335 Pro Val Cys Phe Gly Arg Gln LysAla Pro Val Thr Ala Gly Leu Arg 340 345 350 Tyr Gly Lys Trp 355 12 361PRT Mus musculus 12 Met Asn Asn Phe Ile Leu Leu Glu Glu Gln Leu Ile LysLys Ser Gln 1 5 10 15 Gln Lys Arg Arg Thr Ser Pro Ser Asn Phe Lys ValArg Phe Phe Val 20 25 30 Leu Thr Lys Ala Ser Leu Ala Tyr Phe Glu Asp ArgHis Gly Lys Lys 35 40 45 Arg Thr Leu Lys Gly Ser Ile Glu Leu Ser Arg IleLys Cys Val Glu 50 55 60 Ile Val Lys Ser Asp Ile Ser Ile Pro Cys His TyrLys Tyr Pro Phe 65 70 75 80 Gln Thr Leu Val Tyr Leu Gln Val Val His AspAsn Tyr Leu Leu Tyr 85 90 95 Val Phe Ala Pro Asp Cys Glu Ser Arg Gln ArgTrp Val Leu Thr Leu 100 105 110 Lys Glu Glu Thr Arg Asn Asn Asn Ser LeuVal Ser Lys Tyr His Pro 115 120 125 Asn Phe Trp Met Asp Gly Arg Trp ArgCys Cys Ser Gln Leu Glu Lys 130 135 140 Pro Ala Val Gly Cys Ala Pro TyrAsp Pro Ser Lys Asn Ala Ser Lys 145 150 155 160 Lys Pro Leu Pro Pro ThrPro Glu Asp Asn Arg Arg Ser Phe Gln Glu 165 170 175 Pro Glu Glu Thr LeuVal Ile Ala Leu Tyr Asp Tyr Gln Thr Asn Asp 180 185 190 Pro Gln Glu LeuAla Leu Arg Cys Asp Glu Glu Tyr Tyr Leu Leu Asp 195 200 205 Ser Ser GluIle His Trp Trp Arg Val Gln Asp Lys Asn Gly His Glu 210 215 220 Gly TyrAla Pro Ser Ser Tyr Leu Val Glu Lys Ser Pro Asn Asn Leu 225 230 235 240Glu Thr Tyr Glu Trp Tyr Asn Lys Ser Ile Ser Arg Asp Lys Ala Glu 245 250255 Lys Leu Leu Leu Asp Thr Gly Lys Glu Gly Ala Phe Met Val Arg Asp 260265 270 Ser Arg Thr Pro Gly Thr Tyr Thr Val Ser Val Phe Thr Lys Ala Ile275 280 285 Ile Ser Glu Asn Pro Cys Ile Lys His Tyr His Ile Lys Glu ThrAsn 290 295 300 Asp Ser Pro Lys Arg Tyr Tyr Val Ala Glu Lys Tyr Val PheAsp Ser 305 310 315 320 Ile Pro Leu Leu Ile Gln Tyr His Gln Tyr Asn GlyGly Gly Leu Val 325 330 335 Thr Arg Leu Arg Tyr Pro Val Cys Ser Trp ArgGln Lys Ala Pro Val 340 345 350 Thr Ala Gly Leu Arg Tyr Gly Lys Trp 355360 13 363 PRT Homo sapiens 13 Met Asn Phe Asn Thr Ile Leu Glu Glu IleLeu Ile Lys Arg Ser Gln 1 5 10 15 Gln Lys Lys Lys Thr Ser Pro Leu AsnTyr Lys Glu Arg Leu Phe Val 20 25 30 Leu Thr Lys Ser Met Leu Thr Tyr TyrGlu Gly Arg Ala Glu Lys Lys 35 40 45 Tyr Arg Lys Gly Phe Ile Asp Val SerLys Ile Lys Cys Val Glu Ile 50 55 60 Val Lys Asn Asp Asp Gly Val Ile ProCys Gln Asn Lys Tyr Pro Phe 65 70 75 80 Gln Val Val His Asp Ala Asn ThrLeu Tyr Ile Phe Ala Pro Ser Pro 85 90 95 Gln Ser Arg Asp Leu Trp Val LysLys Leu Lys Glu Glu Ile Lys Asn 100 105 110 Asn Asn Asn Ile Met Ile LysTyr His Pro Lys Phe Trp Thr Asp Gly 115 120 125 Ser Tyr Gln Cys Cys ArgGln Thr Glu Lys Leu Ala Pro Gly Cys Glu 130 135 140 Lys Tyr Asn Leu PheGlu Ser Ser Ile Arg Lys Ala Leu Pro Pro Ala 145 150 155 160 Pro Glu ThrLys Lys Arg Arg Pro Pro Pro Pro Ile Pro Leu Glu Glu 165 170 175 Glu AspAsn Ser Glu Glu Ile Val Val Ala Met Tyr Asp Phe Gln Ala 180 185 190 AlaGlu Gly His Asp Leu Arg Leu Glu Arg Gly Gln Glu Tyr Leu Ile 195 200 205Leu Glu Lys Asn Asp Val His Trp Trp Arg Ala Arg Asp Lys Tyr Gly 210 215220 Asn Glu Gly Tyr Ile Pro Ser Asn Tyr Val Thr Gly Lys Lys Ser Asn 225230 235 240 Asn Leu Asp Gln Tyr Glu Trp Tyr Cys Arg Asn Met Asn Arg SerLys 245 250 255 Ala Glu Gln Leu Leu Arg Ser Glu Asp Lys Glu Gly Gly PheMet Val 260 265 270 Arg Asp Ser Ser Gln Pro Gly Leu Tyr Thr Val Ser LeuTyr Thr Lys 275 280 285 Phe Gly Gly Glu Gly Ser Ser Gly Phe Arg His TyrHis Ile Lys Glu 290 295 300 Thr Thr Thr Ser Pro Lys Lys Tyr Tyr Leu AlaGlu Lys His Ala Phe 305 310 315 320 Gly Ser Ile Pro Glu Ile Ile Glu TyrHis Lys His Asn Ala Ala Gly 325 330 335 Leu Val Thr Arg Leu Arg Tyr ProVal Ser Val Lys Gly Lys Asn Ala 340 345 350 Pro Thr Thr Ala Gly Phe SerTyr Glu Lys Trp 355 360 14 362 PRT Mus musculus 14 Met Asn Phe Asn ThrIle Leu Glu Glu Ile Leu Ile Lys Arg Ser Gln 1 5 10 15 Gln Lys Lys LysThr Ser Leu Leu Asn Tyr Lys Glu Arg Leu Cys Val 20 25 30 Leu Pro Lys SerVal Leu Ser Tyr Tyr Glu Gly Arg Ala Glu Lys Lys 35 40 45 Tyr Arg Lys GlyVal Ile Asp Ile Ser Lys Ile Lys Cys Val Glu Ile 50 55 60 Val Lys Asn AspAsp Gly Val Ile Pro Cys Gln Asn Lys Phe Pro Phe 65 70 75 80 Gln Val ValHis Asp Ala Asn Thr Leu Tyr Ile Phe Ala Pro Ser Pro 85 90 95 Gln Ser ArgAsp Arg Trp Val Lys Lys Leu Lys Glu Glu Ile Lys Asn 100 105 110 Asn AsnAsn Ile Met Ile Lys Tyr His Pro Lys Phe Trp Ala Asp Gly 115 120 125 SerTyr Gln Cys Cys Arg Gln Thr Glu Lys Leu Ala Pro Gly Cys Glu 130 135 140Lys Tyr Asn Leu Phe Glu Ser Ser Ile Arg Lys Thr Leu Pro Pro Ala 145 150155 160 Pro Glu Ile Lys Lys Arg Arg Pro Pro Pro Pro Ile Pro Pro Glu Glu165 170 175 Glu Asn Thr Glu Glu Ile Val Val Ala Met Tyr Asp Phe Gln AlaThr 180 185 190 Glu Ala His Asp Leu Arg Leu Glu Arg Gly Gln Glu Tyr IleIle Leu 195 200 205 Glu Lys Asn Asp Leu His Trp Trp Arg Ala Arg Asp LysTyr Gly Ser 210 215 220 Glu Gly Tyr Ile Pro Ser Asn Tyr Val Thr Gly LysLys Ser Asn Asn 225 230 235 240 Leu Asp Gln Tyr Glu Trp Tyr Cys Arg AsnThr Asn Arg Ser Lys Ala 245 250 255 Glu Gln Leu Leu Arg Thr Glu Asp LysGlu Gly Gly Phe Met Val Arg 260 265 270 Asp Ser Ser Gln Pro Gly Leu TyrThr Val Ser Leu Tyr Thr Lys Phe 275 280 285 Gly Gly Glu Gly Ser Ser GlyPhe Arg His Tyr His Ile Lys Glu Thr 290 295 300 Ala Thr Ser Pro Lys LysTyr Tyr Leu Ala Glu Lys His Ala Phe Gly 305 310 315 320 Ser Ile Pro GluIle Ile Glu Tyr His Lys His Asn Ala Ala Gly Leu 325 330 335 Val Thr ArgLeu Arg Tyr Pro Val Ser Thr Lys Gly Lys Asn Ala Pro 340 345 350 Thr ThrAla Gly Phe Ser Tyr Asp Lys Trp 355 360 15 410 PRT Homo sapiens 15 MetAsp Thr Lys Ser Ile Leu Glu Glu Leu Leu Leu Lys Arg Ser Gln 1 5 10 15Gln Lys Lys Lys Met Ser Pro Asn Asn Tyr Lys Glu Arg Leu Phe Val 20 25 30Leu Thr Lys Thr Asn Leu Ser Tyr Tyr Glu Tyr Asp Lys Met Lys Arg 35 40 45Gly Ser Arg Lys Gly Ser Ile Glu Ile Lys Lys Ile Arg Cys Val Glu 50 55 60Lys Val Asn Leu Glu Glu Gln Thr Pro Val Glu Arg Gln Tyr Pro Phe 65 70 7580 Gln Ile Val Tyr Lys Asp Gly Leu Leu Tyr Val Tyr Ala Ser Asn Glu 85 9095 Glu Ser Arg Ser Gln Trp Leu Lys Ala Leu Gln Lys Glu Ile Arg Gly 100105 110 Asn Pro His Leu Leu Val Lys Tyr His Ser Gly Phe Phe Val Asp Gly115 120 125 Lys Phe Leu Cys Cys Gln Gln Ser Cys Lys Ala Ala Pro Gly CysThr 130 135 140 Leu Trp Glu Ala Tyr Ala Asn Leu His Thr Ala Val Asn GluGlu Lys 145 150 155 160 His Arg Val Pro Thr Phe Pro Asp Arg Val Leu LysIle Pro Arg Ala 165 170 175 Val Pro Val Leu Lys Met Asp Ala Pro Ser SerSer Thr Thr Leu Ala 180 185 190 Gln Tyr Asp Asn Glu Ser Lys Lys Asn TyrGly Ser Gln Pro Pro Ser 195 200 205 Ser Ser Thr Ser Leu Ala Gln Tyr AspSer Asn Ser Lys Lys Ile Tyr 210 215 220 Gly Ser Gln Pro Asn Phe Asn MetGln Tyr Ile Pro Arg Glu Asp Phe 225 230 235 240 Pro Asp Trp Trp Gln ValArg Lys Leu Lys Ser Ser Ser Ser Ser Glu 245 250 255 Asp Val Ala Ser SerAsn Gln Lys Glu Arg Asn Val Asn His Thr Thr 260 265 270 Ser Lys Ile SerTrp Glu Phe Pro Glu Ser Ser Ser Ser Glu Glu Glu 275 280 285 Glu Asn LeuAsp Asp Tyr Asp Trp Phe Ala Gly Asn Ile Ser Arg Ser 290 295 300 Gln SerGlu Gln Leu Leu Arg Gln Lys Gly Lys Glu Gly Ala Phe Met 305 310 315 320Val Arg Asn Ser Ser Gln Val Gly Met Tyr Thr Val Ser Leu Phe Ser 325 330335 Lys Ala Val Asn Asp Lys Lys Gly Thr Val Lys His Tyr His Val His 340345 350 Thr Asn Ala Glu Asn Lys Leu Tyr Leu Ala Glu Asn Tyr Cys Phe Asp355 360 365 Ser Ile Pro Lys Leu Ile His Tyr His Gln His Asn Ser Ala GlyMet 370 375 380 Ile Thr Arg Leu Arg His Pro Val Ser Thr Lys Ala Asn LysVal Pro 385 390 395 400 Asp Ser Val Ser Leu Gly Asn Gly Ile Trp 405 41016 386 PRT Mus musculus 16 Met Glu Ser Lys Ser Ile Leu Glu Glu Leu LeuLeu Lys Lys Ser Gln 1 5 10 15 Gln Lys Lys Lys Met Ser Pro Asn Asn TyrLys Glu Arg Leu Phe Val 20 25 30 Leu Thr Lys Thr Ser Leu Ser Tyr Tyr GluTyr Asp Lys Met Lys Arg 35 40 45 Gly Ser Arg Lys Gly Ser Ile Glu Ile LysLys Ile Arg Cys Val Glu 50 55 60 Lys Val Asn Leu Glu Glu Gln Thr Pro ValGlu Arg Gln Tyr Pro Phe 65 70 75 80 Gln Ile Val Tyr Lys Asp Gly Leu LeuTyr Val Tyr Ala Ser Asn Glu 85 90 95 Glu Ser Arg Cys Gln Trp Leu Lys AlaLeu Gln Lys Glu Ile Arg Gly 100 105 110 Asn Pro His Leu Leu Ile Lys TyrHis Ser Gly Phe Phe Val Asp Gly 115 120 125 Lys Phe Leu Cys Cys Gln GlnSer Cys Lys Ala Ala Pro Gly Cys Thr 130 135 140 Leu Trp Glu Ala Tyr AlaAsp Leu His Ile Ala Ile Ser Asp Glu Lys 145 150 155 160 His Arg Ala ProThr Phe Pro Glu Arg Leu Leu Lys Ile Pro Arg Ala 165 170 175 Val Pro ValLeu Lys Met Asp Ala Ser Ser Ser Gly Ala Ile Leu Pro 180 185 190 Gln TyrAsp Ser Tyr Ser Lys Lys Ser Cys Gly Ser Gln Pro Thr Ser 195 200 205 AsnIle Arg Tyr Ile Pro Arg Glu Asp Cys Pro Asp Trp Trp Gln Val 210 215 220Arg Lys Leu Lys Ser Glu Glu Asp Ile Ala Cys Ser Asn Gln Leu Glu 225 230235 240 Arg Asn Ile Ala Ser His Ser Thr Ser Lys Met Ser Trp Gly Phe Pro245 250 255 Glu Ser Ser Ser Ser Glu Glu Glu Glu Asn Leu His Ala Tyr AspTrp 260 265 270 Phe Ala Gly Asn Ile Ser Arg Ser Gln Ser Glu Gln Leu LeuArg Gln 275 280 285 Lys Gly Lys Glu Gly Ala Phe Met Val Arg Asn Ser SerGln Met Gly 290 295 300 Met Tyr Thr Val Ser Leu Phe Ser Lys Ala Val AsnAsp Lys Lys Gly 305 310 315 320 Thr Val Lys His Tyr His Val His Thr AsnAla Glu Asn Lys Leu Tyr 325 330 335 Leu Ala Glu Asn Tyr Cys Phe Asp SerIle Pro Lys Leu Ile His Tyr 340 345 350 His Gln His Asn Ser Ala Gly MetIle Thr Arg Leu Arg His Pro Val 355 360 365 Ser Thr Lys Ala Asn Lys ValPro Val Ser Val Ala Leu Gly Ser Gly 370 375 380 Ile Trp 385 17 264 PRTHomo sapiens 17 Met Ile Leu Ser Ser Tyr Asn Thr Ile Gln Ser Val Phe CysCys Cys 1 5 10 15 Cys Cys Cys Ser Val Gln Lys Arg Gln Met Arg Thr GlnIle Ser Leu 20 25 30 Ser Thr Asp Glu Glu Leu Pro Glu Lys Tyr Thr Gln HisArg Arg Pro 35 40 45 Trp Leu Ser Gln Leu Ser Asn Lys Lys Gln Ser Asn ThrGly Arg Val 50 55 60 Gln Pro Ser Lys Arg Lys Pro Leu Pro Pro Leu Pro ProSer Glu Val 65 70 75 80 Ala Glu Glu Lys Ile Gln Val Lys Ala Leu Tyr AspPhe Leu Pro Arg 85 90 95 Glu Pro Cys Asn Leu Ala Leu Arg Arg Ala Glu GluTyr Leu Ile Leu 100 105 110 Glu Lys Tyr Asn Pro His Trp Trp Lys Ala ArgAsp Arg Leu Gly Asn 115 120 125 Glu Gly Leu Ile Pro Ser Asn Tyr Val ThrGlu Asn Lys Ile Thr Asn 130 135 140 Leu Glu Ile Tyr Glu Trp Tyr His ArgAsn Ile Thr Arg Asn Gln Ala 145 150 155 160 Glu His Leu Leu Arg Gln GluSer Lys Glu Gly Ala Phe Ile Val Arg 165 170 175 Asp Ser Arg His Leu GlySer Tyr Thr Ile Ser Val Phe Met Gly Ala 180 185 190 Arg Arg Ser Thr GluAla Ala Ile Lys His Tyr Gln Ile Lys Lys Asn 195 200 205 Asp Ser Gly GlnTrp Tyr Val Ala Glu Arg His Ala Phe Gln Ser Ile 210 215 220 Pro Glu LeuIle Trp Tyr His Gln His Asn Ala Ala Gly Leu Met Thr 225 230 235 240 ArgLeu Arg Tyr Pro Val Gly Leu Met Gly Ser Cys Leu Pro Ala Thr 245 250 255Ala Gly Phe Ser Tyr Glu Lys Trp 260 18 264 PRT Mus musculus 18 Met IleLeu Ser Ser Tyr Ser Ser Phe Gln Ser Val Leu Cys Cys Cys 1 5 10 15 CysCys Arg Cys Ser Val Gln Lys Arg Gln Val Arg Thr Gln Ile Ser 20 25 30 LeuSer Arg Glu Glu Glu Leu Ser Glu Lys His Ser Gln Arg Gln Arg 35 40 45 ProTrp Phe Ala Lys Leu Met Gly Lys Thr Gln Ser Asn Arg Gly Gly 50 55 60 ValGln Pro Ser Lys Arg Lys Pro Leu Pro Pro Leu Pro Gln Glu Pro 65 70 75 80Pro Asp Glu Arg Ile Gln Val Lys Ala Leu Tyr Asp Phe Leu Pro Arg 85 90 95Glu Pro Gly Asn Leu Ala Leu Lys Arg Ala Glu Glu Tyr Leu Ile Leu 100 105110 Glu Arg Cys Asp Pro His Trp Trp Lys Ala Arg Asp Arg Phe Gly Asn 115120 125 Glu Gly Leu Ile Pro Ser Asn Tyr Val Thr Glu Asn Arg Leu Ala Asn130 135 140 Leu Glu Ile Tyr Glu Trp Tyr His Lys Asn Ile Thr Arg Asn GlnThr 145 150 155 160 Glu Arg Leu Leu Arg Gln Glu Ala Lys Glu Gly Ala PheIle Val Arg 165 170 175 Asp Ser Arg His Leu Gly Ser Tyr Thr Ile Ser ValPhe Thr Arg Ala 180 185 190 Arg Arg His Thr Gln Ser Ser Ile Lys His TyrGln Ile Lys Lys Asn 195 200 205 Asp Ser Gly Gln Trp Tyr Ile Thr Glu ArgHis Leu Phe Pro Ser Val 210 215 220 Pro Glu Leu Ile Gln Tyr His Gln TyrAsn Ala Ala Gly Leu Ile Ser 225 230 235 240 Arg Leu Arg Tyr Pro Ile GlyLeu Leu Gly Ser Cys Leu Pro Ala Thr 245 250 255 Ser Gly Phe Ser Tyr GluLys Trp 260 19 36 DNA Artificial Sequence Description of ArtificialSequence 3 prime oligo 19 gatcggatcc gaagcttatt ggcgagctca ggattc 36 2043 DNA Artificial Sequence Description of Artificial Sequence 5 primeoligo 20 gatcgcggcc gcaccatggc agcagcacca gtctccacaa gtg 43 21 454 PRTArtificial Sequence Description of Artificial Sequence Translatedpolypeptide sequence of the truncated Btk construct 21 Met Ala Ala AlaPro Val Ser Thr Ser Glu Leu Lys Lys Val Val Ala 1 5 10 15 Leu Tyr AspTyr Met Pro Met Asn Ala Asn Asp Leu Gln Leu Arg Lys 20 25 30 Gly Asp GluTyr Phe Ile Leu Glu Glu Ser Asn Leu Pro Trp Trp Arg 35 40 45 Ala Arg AspLys Asn Gly Gln Glu Gly Tyr Ile Pro Ser Asn Tyr Val 50 55 60 Thr Glu AlaGlu Asp Ser Ile Glu Met Tyr Glu Trp Tyr Ser Lys His 65 70 75 80 Met ThrArg Ser Gln Ala Glu Gln Leu Leu Lys Gln Glu Gly Lys Glu 85 90 95 Gly GlyPhe Ile Val Arg Asp Ser Ser Lys Ala Gly Lys Tyr Thr Val 100 105 110 SerVal Phe Ala Lys Ser Thr Gly Asp Pro Gln Gly Val Ile Arg His 115 120 125Tyr Val Val Cys Ser Thr Pro Gln Ser Gln Tyr Tyr Leu Ala Glu Lys 130 135140 His Leu Phe Ser Thr Ile Pro Glu Leu Ile Asn Tyr His Gln His Asn 145150 155 160 Ser Ala Gly Leu Ile Ser Arg Leu Lys Tyr Pro Val Ser Gln GlnAsn 165 170 175 Lys Asn Ala Pro Ser Thr Ala Gly Leu Gly Tyr Gly Ser TrpGlu Ile 180 185 190 Asp Pro Lys Asp Leu Thr Phe Leu Lys Glu Leu Gly ThrGly Gln Phe 195 200 205 Gly Val Val Lys Tyr Gly Lys Trp Arg Gly Gln TyrAsp Val Ala Ile 210 215 220 Lys Met Ile Lys Glu Gly Ser Met Ser Glu AspGlu Phe Ile Glu Glu 225 230 235 240 Ala Lys Val Met Met Asn Leu Ser HisGlu Lys Leu Val Gln Leu Tyr 245 250 255 Gly Val Cys Thr Lys Gln Arg ProIle Phe Ile Ile Thr Glu Tyr Met 260 265 270 Ala Asn Gly Cys Leu Leu AsnTyr Leu Arg Glu Met Arg His Arg Phe 275 280 285 Gln Thr Gln Gln Leu LeuGlu Met Cys Lys Asp Val Cys Glu Ala Met 290 295 300 Glu Tyr Leu Glu SerLys Gln Phe Leu His Arg Asp Leu Ala Ala Arg 305 310 315 320 Asn Cys LeuVal Asn Asp Gln Gly Val Val Lys Val Ser Asp Phe Gly 325 330 335 Leu SerArg Tyr Val Leu Asp Asp Glu Tyr Thr Ser Ser Val Gly Ser 340 345 350 LysPhe Pro Val Arg Trp Ser Pro Pro Glu Val Leu Met Tyr Ser Lys 355 360 365Phe Ser Ser Lys Ser Asp Ile Trp Ala Phe Gly Val Leu Met Trp Glu 370 375380 Ile Tyr Ser Leu Gly Lys Met Pro Tyr Glu Arg Phe Thr Asn Ser Glu 385390 395 400 Thr Ala Glu His Ile Ala Gln Gly Leu Arg Leu Tyr Arg Pro HisLeu 405 410 415 Ala Ser Glu Lys Val Tyr Thr Ile Met Tyr Ser Cys Trp HisGlu Lys 420 425 430 Ala Asp Glu Arg Pro Thr Phe Lys Ile Leu Leu Ser AsnIle Leu Asp 435 440 445 Val Met Asp Glu Glu Ser 450 22 1365 DNAArtificial Sequence Description of Artificial Sequence Polynucleotidesequence of the truncated Btk construct 22 atggcagcag caccagtctccacaagtgag ctgaaaaagg ttgtggccct ttatgattac 60 atgccaatga atgcaaatgatctacagctg cggaagggtg atgaatattt tatcttggag 120 gaaagcaact taccatggtggagagcacga gataaaaatg ggcaggaagg ctacattcct 180 agtaactatg tcactgaagcggaagactcc atagaaatgt atgagtggta ttccaaacac 240 atgactcgga gtcaggctgagcaactgcta aagcaagagg ggaaagaagg aggtttcatt 300 gtcagagact ccagcaaagctggcaaatat acagtgtctg tgtttgctaa atccacaggg 360 gaccctcaag gggtgatacgtcattatgtt gtgtgttcca cacctcagag ccagtattac 420 ctggctgaga agcaccttttcagcaccatc cctgagctca ttaactacca tcagcacaac 480 tctgcaggac tcatatccaggctcaaatat ccagtgtctc aacaaaacaa gaatgcacct 540 tccactgcag gcctgggatacggatcatgg gaaattgatc caaaggacct gaccttcttg 600 aaggagctgg ggactggacaatttggggta gtgaagtatg ggaaatggag aggccagtac 660 gacgtggcca tcaagatgatcaaagaaggc tccatgtctg aagatgaatt cattgaagaa 720 gccaaagtca tgatgaatctttcccatgag aagctggtgc agttgtatgg cgtctgcacc 780 aagcagcgcc ccatcttcatcatcactgag tacatggcca atggctgcct cctgaactac 840 ctgagggaga tgcgccaccgcttccagact cagcagctgc tagagatgtg caaggatgtc 900 tgtgaagcca tggaatacctggagtcaaag cagttccttc accgagacct ggcagctcga 960 aactgtttgg taaacgatcaaggagttgtt aaagtatctg atttcggcct gtccaggtat 1020 gtcctggatg atgaatacacaagctcagta ggctccaaat ttccagtccg gtggtcccca 1080 ccggaagtcc tgatgtatagcaagttcagc agcaaatctg acatttgggc ttttggggtt 1140 ttgatgtggg aaatttactccctggggaag atgccatatg agagatttac taacagtgag 1200 actgctgaac acattgcccaaggcctacgt ctctacaggc ctcatctggc ttcagagaag 1260 gtatatacca tcatgtacagttgctggcat gagaaagcag atgagcgtcc cactttcaaa 1320 attcttctga gcaatattctagatgtcatg gatgaagaat cctga 1365 23 10 PRT Artificial SequenceDescription of Artificial Sequence Biotinylated peptide 23 Ala Ala AlaGlu Glu Ile Tyr Gly Glu Ile 1 5 10

1. A method for the identification of a compound which modulates theactivity of a Tec kinase polypeptide, comprising contacting (i) atruncated Tec kinase polypeptide wherein the Tec kinase amino acidsequence does not contain the amino acids constituting the PH domain anda portion of the TH domain including at least one proline rich region,but does contain the amino acids constituting the kinase domain with(ii) a test compound, and detecting any enhancement or inhibition in theactivity of the polypeptide, compared to the activity that would occurin the absence of said test compound.
 2. A method as claimed in claim 1wherein the Tec kinase amino acid sequence contains the amino acidsconstituting the SH2 and kinase domain.
 3. A method as claimed in claim1 wherein the Tec kinase amino acid sequence contains the amino acidsconstituting the SH3, SH2 and kinase domain.
 4. A method according toclaim 1 wherein the truncated Tec kinase polypeptide does not containany proline rich regions.
 5. A method according to claim 1 wherein theTec kinase polypeptide is Itk.
 6. A method as claimed in claim 5 whereinthe Tec kinase polypeptide has an amino acid sequence selected from thesequence of FIG. 3 and sequences having at least 70% homology thereto.7. A method as claimed in claim 1 wherein the Tec kinase polypeptide isBtk.
 8. A method as claimed in claim 7 wherein the Tec kinasepolypeptide has an amino acid sequence selected from the sequence setforth in FIG. 8 and sequences having at least 70% homology thereto.
 9. Amethod for the identification of a compound as claimed in claim 1,comprising contacting a compound of interest with the truncated Teckinase polypeptide in the presence of a target polypeptide and observingany enhancement or inhibition of phosphorylation of the targetpolypeptide.
 10. A method according to claim 1 wherein a cell lineexpressing the truncated Tec kinase polypeptide is contacted with thetest compound.
 11. A truncated Tec kinase polypeptide having a Itkkinase amino acid sequence that does not contain the amino acidsconstituting the PH domain and a portion of the TH domain including atleast one proline rich region, but does contain the amino acidsconstituting the kinase domain.
 12. A truncated Tec kinase polypeptidehaving a Tec kinase amino acid sequence that does not contain the aminoacids constituting the PH domain and a portion of the TH domainincluding at least one proline rich region, but does contain the aminoacids constituting the SH2 and kinase domain.
 13. A truncated Tec kinasepolypeptide as claimed in claim 12 wherein the Tec kinase amino acidsequence contains the amino acids constituting the SH3, SH2 and kinasedomain.
 14. A truncated Tec kinase polypeptide as claimed in claim 12which does not contain any proline-rich regions.
 15. A truncated Teckinase polypeptide as claimed in claim 11, which does not contain anyproline-rich regions.
 16. A truncated Tec kinase polypeptide as claimedin claim 11 having an amino acid sequence selected from (a) the sequenceset forth in FIG. 3 and (b) sequences having at least 70% homologythereto.
 17. A turmcated Tec kinase polypeptide as claimed in claim 12wherein the Tec kinase polypeptide is Btk.
 18. A truncated Tec kinasepolypeptide as claimed in claim 17 having an amino acid sequenceselected from (a) the sequence set forth in FIG. 8 and (b) sequenceshaving at least 70% homology thereto.
 19. An isolated polynucleotidewhich: (a) encodes a truncated Tec kinase polypeptide according to claim1; (b) is complementary to polynucleotide (a); (c) selectivelyhybridises to polynucleotide (a) or (b); or (d) is degenerate as aresult of the genetic code from polynucleotide (a), (b) or (c).
 20. Anisolated polynucelotide as claimed in claim 19 which: (a) encodes thetruncated Itk polypeptide set forth in FIG. 3; (b) is complementary topolynucleotide (a); (c) selectively hybridises to polynucleotide (a) or(b); or (d) is degenerate as a result of the genetic code frompolynucleotide (a), (b) or (c).
 21. An isolated polynucleotide asclaimed in claim 20 having: (a) the sequence set forth in FIG. 2; (b) asequence complementary to polynucleotide (a); (c) a sequence whichselectively hybridises to polynucleotide (a) or (b); or (d) a sequencethat is degenerate as a result of the genetic code from polynucleotide(a), (b) or (c).
 22. An isolated polynucelotide as claimed in claim 19which: (a) encodes the truncated Btk polypeptide set forth in FIG. 8;(b) is complementary to polynucleotide (a); (c) selectively hybridisesto polynucleotide (a) or (b); or (d)is degenerate as a result of thegenetic code from polynucleotide (a), (b) or (c).
 23. An isolatedpolynucleotide as claimed in claim 22 having: (a) the sequence set forthin FIG. 9; (b) a sequence complementary to polynucleotide (a); (c) asequence which selectively hybridises to polynucleotide (a) or (b); or(d) a sequence that is degenerate as a result of the genetic code frompolynucleotide (a), (b) or (c).
 24. A vector comprising a polynucleotideaccording to claim
 19. 25. A host cell comprising a vector as claimed inclaim
 24. 26. (Cancel)
 27. A method of producing a polypeptide, whichmethod comprises introducing into an appropriate cell line a vectorcomprising a polynucleotide as claimed in claim 9 under conditionssuitable for obtaining expression of the polypeptide encoded by saidpolynucleotide.
 28. (Cancel)
 29. (Cancel)
 30. (Cancel)
 31. (Cancel)